DESCRIPTION: (provided by the applicant): Giardia lamblia is one of the most prevalent parasitic protists and a major cause of diarrheal disease worldwide. A parasitic lifestyle and an extremely early divergence from the eukaryotic tree have permitted Giardia to develop independent solutions to common eukaryotic cellular problems. This biological diversity is instrumental in their ability to cause disease. Notable features of Giardia biology are its two nuclei and its unusually compact and plastic genome. Our long-term goal is to understand the consequences that genomic structure exerts on gene expression. A fundamental starting point is the study of transcriptional regulation. Although 'rules' of transcription have been delineated in higher eukaryotes, extensive work in lower eukaryotes and archaea have clearly indicated many differences in the mechanics of gene expression. Investigation of transcription in Giardia is therefore important both as a prerequisite to understand expression of proteins involved in pathogenesis and also to further our understanding of the evolution of transcriptional machinery. We will test two hypotheses regarding RNA Polymerase II transcription in Giardia lamblia: (I) Giardia will contain a simplified and degenerate core promoter. Two core promoter elements, the TATA box and initiator element (Inr), will be present, although based on their similar sequence profiles, they will be independently able to initiate transcription. A homologue of TATA binding protein will be present to direct transcription from both the TATA box and mnr. (II) The relaxed sequence constraints for transcription initiation and the presence of a tetraploid genome distributed between two nuclei suggests an enhanced role for gene regulation at the chromatin and nuclear level. To experimentally test these hypotheses we will ronduct four specific aims: (1) Characterization of the roles served by core promoter elements in our model promoter - alpha-2 tubulin. (2) Characterization of cryptic promoter elements. (3) Functional rharacterization of a homologue of TATA binding protein (TBP). (4) Examination of transcriptional regulation at the chromatin and nuclear level. These studies will build an understanding of transcription in Giardia lamblia and lay foundations for study of the role of the regulation of gene expression in disease.